Why am I unable to see phalloidin staining in my methanol-fixed and permeabilized samples?

Phalloidin is only able to bind the native quaternary structure of F-actin. In order to retain the quaternary protein structure, formaldehyde fixation should be utilized. Methanol and other alcohols denature the native conformation and are therefore not compatible with phalloidin staining.

If your staining conditions require the use of methanol or other alcohols, we recommend an antibody-based approach to visualize the actin cytoskeleton.  We have found that our β-Actin (8H10D10) Mouse mAb #3700 and β-Actin (13E5) Rabbit mAb #4970 work very well in methanol-fixed or permeabilized samples.
 

Last updated: February 27, 2024